TGF-β/Smad2 signalling regulates enchondral bone formation of Gli1+ periosteal cells during fracture healing

Abstract

Objectives: Most bone fracture heals through enchondral bone formation that relies on the involvement of periosteal progenitor cells. However, the identity of periosteal progenitor cells and the regulatory mechanism of their proliferation and differentiation remain unclear. The aim of this study was to investigate whether Gli1-Cre^ERT2 can identify a population of murine periosteal progenitor cells and the role of TGF-β signalling in periosteal progenitor cells on fracture healing.

Materials and methods: Double heterozygous Gli1-CreER^T2 ;Rosa26-tdTomato^flox/wt mice were sacrificed at different time points for tracing the fate of Gli1⁺ cells in both intact and fracture bone. Gli1-CreER^T2 -mediated Tgfbr2 knockout (Gli1-CreER^T2 ;Tgfbr2^flox/flox ) mice were subjected to fracture surgery. At 4, 7, 10, 14 and 21 days post-surgery, tibia samples were harvested for tissue analyses including μCT, histology, real-time PCR and immunofluorescence staining.

Results: Through cell lineage-tracing experiments, we have revealed that Gli1-Cre^{ER T2} can be used to identify a subpopulation of periosteal progenitor cells in vivo that persistently reside in periosteum and contribute to osteochondral elements during fracture repair. During the healing process, TGF-β signalling is continually activated in the reparative Gli1⁺ periosteal cells. Conditional knockout of Tgfbr2 in these cells leads to a delayed and impaired enchondral bone formation, at least partially due to the reduced proliferation and chondrogenic and osteogenic differentiation of Gli1⁺ periosteal cells.

Conclusions: TGF-β signalling plays an essential role on fracture repair via regulating enchondral bone formation process of Gli1⁺ periosteal cells.

Keywords: Gli1; TGF-β signalling; enchondral bone formation; fracture healing; periosteum.

Figure 1

Postnatal Gli1⁺ cells reside in periosteum. Tomato^Gli1ER mice were administered with tamoxifen at 1 mo of age and analysed at 1, 3, 6 and 12 mo later. A‐D, Representative images of tibiae from each time point mapped Gli1⁺ cells evidently in articular cartilage, growth plate, chondro‐osseous junction and periosteum. (a‐d) ABH stained images of tibiae were an adjacent section to (A‐D), respectively, and (a‐d) indicated the expression locations of Gli1⁺ cells at each time point in (A‐D). E‐H, The numbers of Gli1⁺ cells in periosteal area of the diaphyseal cortical bones. CB: cortical bone, BM: bone marrow. Red: tdTomato⁺ cells, blue: nuclear staining by DAPI. Scale bars: 1000 µm

Figure 2

Gli1⁺ periosteal cells undergo proliferation and differentiation into chondrocytes, osteoblasts and osteocytes during fracture healing. Tomato^Gli1ER mice induced with tamoxifen at 1 mo of age were subjected to the fracture surgery at 10 wk of age and sacrificed 4, 7, 10, 14 and 35 d later. A‐E, Representative immunofluorescence images of fractured tibiae from each time point. F‐J, High magnification images of local fracture sites in (A‐E), respectively. (f‐j) ABH stained images were an adjacent section to (F‐J) respectively, indicating the components that Gli1⁺ cells proliferated and differentiated at each time point. (A, F, f) Gli1⁺ cells largely expanded on the periosteal surface closed to the fracture site at day 4. Yellow arrows: expanded periosteum. B‐D, G‐I, g‐i, Gli1⁺ cells differentiated into chondrocytes, osteoblasts and osteocytes to form fracture callus at days 7‐14. Red arrows: chondrogenic differentiated Gli1⁺ cells. Green arrows: osteogenic differentiated Gli1⁺ cells. (E, J, j) Gli1⁺ cells were presented in the newly formed periosteum at day 35. K, Schematic experimental design for data in (L). L, Gli1⁺ periosteal cells both near to and away from the fracture sites highly expressed immunofluorescence signal of Cidu (green) at day 4 post‐fracture. Red: tdTomato⁺ cells, blue: nuclear staining by DAPI. Scale bars: 1000 µm

Figure 3

Periosteal‐derived Gli1⁺ cells are essential for fracture healing. Tomato^Gli1ER mice induced with tamoxifen at 1 mo of age were subjected to tibia fracture surgery combined with removing antero‐ and posterior‐lateral periosteum at 10‐wk‐old. A, Representative three‐dimensional (3D) µCT images showed a distinct fracture line (red arrow) at day 14 in the periosteum removed mice. B, C, Quantitative μCT analysis of bone volume and BV/TV for fracture callus tissues at day 14. D‐G, No Gli1⁺ cells were presented in the periosteum removed side at days 4 and 14 post‐fracture. (d‐g) ABH staining of an adjacent section to (D‐G), respectively. The periosteum removed mice presented a deficiency of periosteal expansion at day 4 (black arrows) and almost no bone callus formation (red arrows) at day 14

Figure 4

TGF‐β/Smad2 signalling is activated in Gli1⁺ periosteal cells during fracture healing. A, Immunofluorescence signal of TGF‐β1 (green) in the uninjured cortical bone, fracture haematoma at day 4, cartilage matrix at day 7 and bone matrix at day 14. B, Immunofluorescence signal of p‐Smad2 (green) in the uninjured Gli1⁺ periosteal cells, the expending Gli1⁺ periosteal cells at day 4, the chondrogenic differentiated Gli1⁺ cells at day 7 and the osteogenic differentiated Gli1⁺ cells at day 14. C, Percentage of TGF‐β1⁺ area quantified in the respective regions at different time points. D, Percentage of p‐Smad2⁺ tdTomato⁺ over tdTomato⁺ cells quantified in the respective regions at different time points. CB: cortical bone, BM: bone marrow, Red: tdTomato⁺ cells, blue: nuclear staining by DAPI. Scale bars: 1000 µm

Figure 5

TGF‐β neutralizing antibody results in a delayed enchondral bone formation in fractured mice. TGF‐β neutralizing antibody (5 mg/kg body, once every 2 d) was subcutaneously injected into fractured regions immediately after fracture. A, Representative µCT images showed a distinct fracture line at day 21 (red arrow) in TGF‐β neutralizing antibody administrated mice. B, C, Quantitative μCT analysis of callus bone volume (BV) and callus mineralized volume fraction (BV/TV) at different time points. D, ABH staining of fracture callus at different time points. Dotted lines: the expanding periosteum, black arrows: cartilage area, yellow arrow: woven bone area. E, Percentage of cartilage area over periosteal callus area (Cg.Ar/Ps.Cl.Ar, %). F, Percentage of woven bone area over periosteal callus area (Md.Ar/Ps.Cl.Ar, %). Scale bars: 1000 µm

Figure 6

Deletion of Tgfbr2 in Gli1⁺ periosteal cells leads to a delayed and impaired enchondral bone formation in fractured mice. Tgfbr2^Gli1ER mice induced with tamoxifen at 1 mo of age were subjected to tibial fracture surgery at 10 wk of age. A, Representative µCT images showed the distinct fracture lines at day 14 and 21 (red arrow) in Tgfbr2^Gli1ER mice compared to Cre‐negative mice. B, C, Quantitative μCT analysis of callus bone volume (BV) and callus mineralized volume fraction (BV/TV) at different time points. D, ABH staining of fracture callus at different time points. Dotted lines: the expanding periosteum, black arrows: cartilage area, yellow arrow: woven bone area. E, Percentage of cartilage area over periosteal callus area (Cg.Ar/Ps.Cl.Ar, %). F, Percentage of woven bone area over periosteal callus area (Md.Ar/Ps.Cl.Ar, %). Scale bars: 1000 µm

Figure 7

Deletion of Tgfbr2 in Gli1⁺ periosteal cells down‐regulates expressions of chondrocyte‐ and osteoblast‐specific marker genes in callus tissues. Total RNA was extracted from callus tissues (n = 3) of Tgfbr2^Gli1ER mice at different time points. A, Expression of Tgfbr2 was decreased at day 7‐21. B, Expression of Col2a1 was decreased at day 7. C, Expression of Col10a1 was decreased at day 7 and 10, but increased at day 14. D, E, Expression of Runx2 and osteocalin was decreased at day 10 and 14, but increased at day 21

Figure 8

Deletion of Tgfbr2 in Gli1⁺ periosteal cells inhibits proliferation and differentiation of Gli1⁺ periosteal cells into chondrocytes and osteoblasts during fracture healing. Tgfbr2^Gli1ER;ROSA^tdTomato mice induced with tamoxifen at 1 mo of age were subjected to fracture surgery at 10 wk of age. A, Percentage of tdTomato⁺; Cidu⁺ over tdTomato⁺ cells. B, Percentage of tdTomato⁺ chondrocytes surrounded by green fluorescence signal of Col‐II over tdTomato⁺ cells. C, Percentage of tdTomato⁺; Cidu⁺ over tdTomato⁺ cells. Scale bars: 1000 µm